Animal integration vector and methods for its use

Abstract

Novel P element containing vectors and methods for their use to insert an exogenous nucleic acid into the genome of an animal are provided. The subject vectors have a pair of P element transposase recognized insertion sites, e.g. 31 base pair inverted repeats, flanking at least one transcriptionally active gene that is in proximity to at least one of the P element transposase recognized sites. In practicing the subject methods, a vector of the subject invention is introduced into the animal cell under conditions sufficient for transposition to occur. The subject methods find use in a variety of applications in which the insertion of an exogenous nucleic acid into the genome of an animal is desired, e.g. include research, synthesis and therapeutic applications.

1 . A P element vector that integrates into the genome of a non-Drosophilidae animal, said vector comprising: a pair of P element transposase recognized insertion sequences flanking at least one transcriptionally active gene that is in close approximation to one of the P element transposase recognized sequences.  
     
     
         2 . The vector according to  claim 1 , wherein said at least one transcriptionally active gene comprises a coding sequence that is expressed under intracellular conditions.  
     
     
         3 . The vector according to  claim 1 , wherein said vector further comprises at least one endonuclease cleavage site.  
     
     
         4 . The vector according to  claim 1 , wherein said endonuclease cleavage site is present in a polylinker.  
     
     
         5 . The vector according to  claim 1 , wherein said vector further comprises transposase domain encoding a product having P element transposase activity, wherein said transposase domain is not flanked by said pair of transposase recognized insertion sequences.  
     
     
         6 . The vector according to  claim 1 , wherein said vector further comprises an exogenous sequence positioned at a site between said pair of transposase recognized insertion sequences.  
     
     
         7 . The vector according to  claim 1 , wherein said transposase recognized insertion sequences are 31 base pair inverted repeats.  
     
     
         8 . A P element vector for introducing an exogenous nucleic acid into the genome of a non-Drosophilidae animal, said vector comprising: a pair of P element derived 31 base pair inverted repeats flanking at least one transcriptionally active gene, wherein said transcriptionally active gene is in proximity of at least one of the P element 31 base pair inverted repeats and comprises a coding sequence that is expressed under intracellular conditions.  
     
     
         9 . The vector according to  claim 8 , wherein said vector further comprises a nucleic acid sequence encoding a product having P element transposase activity positioned external to the vector domain flanked by said pair of P element derived 31 base pair inverted repeats.  
     
     
         10 . The vector according to  claim 8 , wherein said vector further comprises an exogenous nucleic acid positioned between said P element derived 31 base pair inverted repeats.  
     
     
         11 . A method of inserting an exogenous nucleic acid into the genome of a non-Drosophilidae animal, said method comprising: 
 introducing into said animal a P-element derived vector comprising said exogenous nucleic acid under conditions sufficient for transposition to occur;    whereby said exogenous nucleic acid is inserted into said genome.    
     
     
         12 . A method of inserting an exogenous nucleic acid into the genome of a non-Drosophilidae animal, said method comprising: 
 introducing into said animal a vector according to  claim 1  under conditions sufficient for transposition to occur;    whereby said exogenous nucleic acid is inserted into said genome.    
     
     
         13 . The method according to  claim 12 , wherein said vector comprises a transposase domain.  
     
     
         14 . The method according to  claim 12 , wherein said method further comprises introducing a second vector comprising a transposase domain into said animal.  
     
     
         15 . The method according to  claim 12 , wherein said exogenous nucleic acid ranges in length from about 50 to 150,000 bp.  
     
     
         16 . The method according to  claim 12 , wherein said target animal is a vertebrate.  
     
     
         17 . The method according to  claim 12 , wherein said vertebrate animal is a mammalian animal.  
     
     
         18 . The method according to  claim 12 , wherein said mammalian animal is a rodent.  
     
     
         19 . A kit for use in inserting an exogenous nucleic acid into a target cell, said kit comprising: 
 a P element derived vector comprising a pair of P element transposase recognized insertion sequences flanking at least one transcriptionally active gene in proximity to at least one of the P element transposase recognized isertion sequences.    
     
     
         20 . The kit according to  claim 19 , wherein said transcriptionally active gene comprises a coding sequence that is expressed under intracellular conditions.  
     
     
         21 . The kit according to  claim 19 , wherein said vector further comprises at least one endonuclease cleavage site positioned between said transposase recognized insertion sequences.  
     
     
         22 . The kit according to  claim 21 , wherein said endonuclease cleavage site is present in a polylinker.  
     
     
         23 . The kit according to  claim 19 , wherein said kit further comprises a nucleic acid sequence encoding a product having P element transposase activity.  
     
     
         24 . The kit according to  claim 23 , wherein said vector comprises said nucleic acid sequence encoding a product having transposase activity.  
     
     
         25 . The kit according to  claim 23 , wherein said nucleic acid sequence encoding a product having transposase activity is present on a second vector.  
     
     
         26 . The kit according to  claim 19 , wherein said transposase recognized insertion sequences are 31 base pair inverted repeats.  
     
     
         27 . A non-Drosophilidae animal or cells derived from said animal that has P element transposase recognized insertion sequences integrated into the genome.  
     
     
         28 . The animal or cells according to  claim 27 , wherein said animal is a vertebrate or said cells are vertebrate cells.  
     
     
         29 . The animal or cells according to  claim 28 , wherein said animal is a mammal or said cells are mammalian cells.  
     
     
         30 . The animal or cells according to  claim 29 , wherein said animal is a rodent or said cells are rodent cells.  
     
     
         31 . A non-Drosophilidae animal or cells derived from said animal that have P element transposase recognized 31 bp insertion sequences integrated into the genome.  
     
     
         32 . The animal or cells according to  claim 31 , wherein said animal is a vertebrate or said cells are vertebrate cells.  
     
     
         33 . The animal or cells according to  claim 32 , wherein said animal is a mammal or said cells are mammalian cells.  
     
     
         34 . The animal or cells according to  claim 33 , wherein said animal is a rodent or said cells are rodent cells.